Human .alpha..sub.2 -macroglobulin (.alpha..sub.2 M) is a 718-kDa glycoprotein that was originally characterized as a broad-spectrum proteinase inhibitor. More recent experiments have demonstrated that (.alpha..sub.2 -Macroglobulin (.alpha..sub.2 M) also binds and regulates the activity of various cytokines, see Gonias, Exp. Hematol. 20:302-311 (1992). The structure of .alpha..sub.2 M consists of four identical subunits, each with 1451 amino acids. The subunits are linked into dimers by disulfide bonds and into intact homotetramers by noncovalent interactions.
Proteinases react with .alpha..sub.2 M by cleaving any of a number of susceptible peptide bonds in the "bait region", which includes amino acids 666-706. Bait region cleavage causes .alpha..sub.2 M to undergo a major conformational change, which effectively "traps" the attacking proteinase in a complex which is non-dissociable, even when the proteinase and the inhibitor are not covalently linked. Conformational change also reveals binding sites for the .alpha..sub.2 M receptor/low density-lipoprotein receptor-related protein (LRP). These binding sites have been localized to 18-kDa peptides at the C-terminus of each .alpha..sub.2 M subunit; Lys-1370 and Lys-1374 play particularly important roles.
Like the complement components, C3 and C4, each .alpha..sub.2 M subunit contains a novel thiol ester bond, which is formed from the side-chains of Cys-949 and Glu-952, and these thiol esters may be instrumental in determining the conformational state of .alpha..sub.2 M. When .alpha..sub.2 M reacts with a proteinase, the thiol esters emerge from within hydrophobic, solvent-restricted clefts and are cleaved by nucleophiles or H.sub.2 O. Small primary amines, such as methylamine, penetrate the hydrophobic clefts and react with .alpha..sub.2 M thiol esters independently of proteinases, inducing an equivalent or nearly equivalent conformational change.
In addition to its activity as a proteinase inhibitor, .alpha..sub.2 M functions as a major carrier and regulator of certain cytokines, including isoforms of the transforming growth factor-.beta. (TGF-.beta.) family. The highest affinity interactions of .alpha..sub.2 M involve members of the transforming growth factor-.beta. (TGF-.beta.) and neurotorphin families. O'Connor-McCourt and Wakefield first identified .alpha..sub.2 M as a physiologically significant carrier of TGF-.beta. in human serum (J. Biol. Chem. 262, 14090-14099, 1987). Their studies demonstrated that nearly all of the TGF-.beta.1 in serum is associated with .alpha..sub.2 M and that the bound TGF-.beta.1 is inactive. Huang et al. (J. Biol. Chem. 263, 1535-1541, 1988) confirmed the role of .alpha..sub.2 M as a TGF-.beta.-carrier and demonstrated that the TGF-.beta.-binding activity of .alpha..sub.2 M depends on its conformational state.
More recent studies have demonstrated the function of .alpha..sub.2 M as a TGF-.beta.-carrier in animal model systems. When radioiodinated TGF-.beta.1 is injected intravascularly in mice, the cytokine is cleared rapidly at first; however, this is followed by a slow-clearance phase, during which time the TGF-.beta. is almost entirely .alpha..sub.2 M-associated.
The TGF-.beta. family of cytokines regulates diverse processes including cellular growth, differentiation, wound healing, and inflammation. At the cellular level, TGF-.beta. response is mediated by or regulated by a variety of receptors and binding proteins, including the type I and type II receptors, which are serine/threonine kinases, betaglycan, and endoglin. TGF-.beta. activity is also regulated by processes that alter delivery of the active cytokine to the cell surface. For example, TGF-.beta. is secreted as a large latent complex that includes the propeptide, latency associated peptide (LAP), and a second gene product, latent TGF-.beta.-binding protein (LTBP). Conversion of latent TGF-.beta. into active 25-kDa homodimer requires dissociation of LAP and LTBP in reactions which may be mediated by proteinases, thrombospondin, the mannose 6-phosphate/insulin-like growth factor-II receptor and acidic microenvirornents. Once activated, the 25-kDa form of TGF-.beta. may bind to .alpha..sub.2 M, once again forming a complex that is unavailable for receptor-binding.
Binding of TGF-.beta. to .alpha..sub.2 M is initially non-covalent and reversible; however, the complex can become covalently stabilized as a result of thiol-disulfide exchange. The latter reaction is observed primarily with conformationally-altered .alpha..sub.2 M, since native .alpha..sub.2 M lacks free thiol groups. A number of complementary methods have been used to determine equilibrium dissociation constants (K.sub.D) for the interaction of TGF-.beta. with .alpha..sub.2 M (Arch. Biochem. Biophys. 292,487-49,1992;J. Biol. Chem. 269,1533-1540, 1994; and Ann. N. Y. Acad. Sci. USA 737,273-290, 1994). The K.sub.D s for the binding of TGF-.beta.1 and TGF-.beta.2 to native .alpha..sub.2 M are 300 nNM and 10 nM, respectively; the K.sub.D s for the binding of TGF-.beta.1 and TGF-.beta.2 to methylamine-modified .alpha..sub.2 M (.alpha..sub.2 M-MA) are 80 nM and 10 nM, respectively. These binding constants accurately predict the ability of .alpha..sub.2 M to neutralize TGF-.beta. in cell culture systems.
In cell culture systems, .alpha..sub.2 M neutralizes both exogenously-added and endogenously-synthesized TGF-.beta.. Neutralization of endogenously-synthesized TGF-.beta. results in altered gene expression, including greatly increased expression of inducible nitric oxide synthase (iNOS) by murine macrophages and increased expression of platelet-derived growth factor .alpha.-receptor by vascular smooth muscle cells (see J. Biol. Chem. 270, 21919-21927, 1995 and J. Biol. Chem. 270, 30741-30748, 1995). .alpha..sub.2 M gene knock-out mice demonstrate increased tolerance to endotoxin challenge and this characteristic is most likely explained by the enhanced function of TGF-.beta. as an immunosuppressive, in the absence of .alpha..sub.2 M.
The fate of .alpha..sub.2 M-associated TGF-.beta. depends on the .alpha..sub.2 M conformation. Native .alpha..sub.2 M, which is the predominant form of .alpha..sub.2 M present in the plasma and probably in most extravascular microenvironments, binds TGF-.beta. reversibly and noncovalently. Thus, native .alpha..sub.2 M may buffer tissues against rapid changes in TGF-.beta. levels by binding or slowly releasing the cytokine in response to the free TGF-.beta. concentration. Based on the K.sub.D value, it is believed that approximately 95% of the TGF-.beta.1 in plasma is .alpha..sub.2 M-associated under equilibrium conditions, even though TGF-.beta.1 binds to native .alpha..sub.2 M with lower affinity than TGF-.beta.2. Conversion of .alpha..sub.2 M into the transformed conformation, which probably occurs most frequently at sites of inflammation due to the increase in cellular proteinase secretion, alters the mechanisms by which TGF-.beta. is regulated. First, transformed .alpha..sub.2 M has free Cys residues and thus undergoes thiol-disulfide exchange with TGF-.beta., eliminating the potential for release of active cytokine. Second, .alpha..sub.2 M-proteinase complexes bind to the endocytic receptor, LRP; bound TGF-.beta. is internalized with the .alpha..sub.2 M-proteinase complex and probably delivered to lysosomes.
The mechanism by which .alpha..sub.2 M binds cytokines remains unclear. Early studies, suggesting a prominent role for the thiol ester-derived Cys-residues, were not confirmed for TGF-.beta.1 and TGF-.beta.2. When .alpha..sub.2 M-MA was treated with papain to release the 18-kDa receptor binding domains, the TGF-.beta.-binding activity remained with the residual 600-kDa .alpha..sub.2 M fragment. Thus, the cytokine- and LRP-binding sites are not co-localized. One proposed mechanism for .alpha..sub.2 M binding was that the central cavity in the structure of .alpha..sub.2 M, which serves as the proteinase-trap, also non-specifically binds cytokines. Arguments in support of the this model include: the complex quaternary structure of .alpha..sub.2 M, the known trapping mechanism by which .alpha..sub.2 M interacts with proteinases, and the large number of structurally unrelated cytokines which have been reported to associate with .alpha..sub.2 M. In accordance with the present invention the TGF-.beta.-binding domain has now been localized to a specific region in the structure of human .alpha..sub.2 M.